The major focus of this project is to elucidate the mechanisms controlling cell fate decisions in developing T cells. Precursor T cells undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive T cell antigen receptors (TCR) do not mature (positive and negative selection). These selection processes require TCR engagement of self-MHC antigens, but different aspects of these interactions determine whether the cells will live or die. TCR signals also promote other differentiation events, as well as the development of lineages. Early precursor thymocytes that commit to the T cell lineage must specify an alpha-beta (ab) or gamma-delta (gd) T cell fate. With the appropriate TCR-MHC interactions, T cells developing in the ab pathway will adopt a CD4 helper or CD8 cytotoxic T cell fate. A major goal in our work is to understand how TCR signals, acting in concert with other developmental cues, are linked to the process of lineage commitment. In our previous work, we have shown that quantitative differences in TCR signaling can instruct many cell fate decisions in developing thymocytes. We have found that deficiencies of certain kinases, required for TCR activation, favor a CD8 over a CD4 fate. Moreover, limiting thymocyte migration and the associated interactions between TCR and MHC also favors the CD8 fate, indicating that the kinetics of TCR signaling could play a role in lineage commitment. Similarly, we find that quantitative differences in TCR signaling influence the gd versus ab lineage decision. Mutations that enhance TCR signaling favor gd at the expense of ab lineage development. Signals through the highly conserved transmembrane receptor, Notch, also influence cell fate decisions in developing T cells. We find that a constitutively active form of Notch can override the bias normally imposed by specific TCR signals, suggesting that these two signaling systems may act in concert to specify cell fate. In some systems, Notch activity can be regulated by ligand binding or by association with other proteins like Numb and Fringe that inhibit responses to Notch ligands. In this regard, we observe that an isoform of Numb is differentially regulated in developing CD4 and CD8 thymocytes. Since Numb has the potential to interact with components of the TCR signaling pathway, this protein is a prime candidate for linking TCR signals to Notch. In other efforts to understand how Notch signaling is regulated in the thymus, we have studied Presenilins, proteins that are required for generating the active form of Notch. We have developed a novel system for assessing Notch function in vivo that gets around the problems of redundancy in Notch receptors/ligands, early lethality associated with deletion mutants, and those associated with ectopic and over expression of transgenes. We have used a transgene, expressing a dominant negative form of presenilin, which produces a profound block in T cell development, while promoting the ectopic development of B cells. This protein appears to act predominantly in an early thymic precursor, also enhancing NK development while attenuating gd development. It is most likely that this dominant negative form of Presenilin is acting through Notch since these functional defects can be compensated by the co-expression of a transgene encoding an active form of Notch. This belief is further supported by our findings that Amyloid Precursor Protein (APP) family members (the other major substrate for Presenilin-mediated activity) are not expressed by thymocytes and that deletion mutants of these proteins have no effect on thymic development. Thus, Notch appears to regulate lineage decisions at several stages of thymic development, including T/B lineage commitment.